ABSTRACT: Renal ischemia/reperfusion (I/R) is a major problem leading to kidney damage following renal transplantation or major vascular surgery. Our laboratory has demonstrated that the major antioxidant in the mitochondria, manganese superoxide dismutase (MnSOD), is inactivated during renal transplantation (human and rodent) and renal I/R. These data suggested that the loss of MnSOD activity may be one key event that results in subsequent renal dysfunction, which is supported by preliminary data showing that induction of MnSOD (via gene delivery and estradiol pretreatment) protects the kidney from I/R injury. Conversely, compelling new data show that downregulation of MnSOD (using MnSOD heterozygote (-/+) transgenic mice) results in augmentation of mitochondrial and renal injury. Inactivation of MnSOD results in mitochondrial generation of superoxide and presumably mitochondrial damage; however, the mechanistic pathways involved with this injury remain unknown. Exciting new studies which focused on the five mitochondrial electron transport complexes, revealed alterations in Complexes III, IV, and V following renal I/R, which would also contribute to mitochondrial oxidant production. Thus, we hypothesize that: Electron transport complexes are targets of mitochondrial oxidant damage during I/R and that damage to specific complexes are the critical downstream event(s) that result from inactivation of MnSOD. We will use novel transgenic mouse models and renal cells designed to bi- directionally modulate MnSOD expression, along with cutting-edge proteomic analysis that will lead to identification of key mitochondrial targets that play a fundamental role in injury following renal I/R. Hypothesis 1. Even modest reductions in MnSOD activity (partial knockdown) lead to mitochondrial complex damage due to increased oxidant production following renal I/R. To test this hypothesis, MnSOD knockdown (using siRNA technology and mutant mice) will be combined with measurements of oxidant generation, mitochondrial integrity, cell viability, renal function, and mitochondrial proteomic analyses to determine the precise targets (complexes and/or subunits of complexes) and pathways involved with mitochondrial complex damage following MnSOD knockdown and I/R. Hypothesis 2. Increased MnSOD activity reduces oxidant production, restores normal mitochondrial complex function, and blunts renal injury following I/R. To test this hypothesis, MnSOD overexpression (using gene delivery, transgenic mice, and estradiol-mediated induction) will be combined with measurements of oxidant generation, cell viability, renal function, and mitochondrial proteomic analyses to determine the mechanisms that mediate protection from I/R injury due to MnSOD induction. Hypothesis 3. The new generation catalytic antioxidant manganese porphyrin (MnP) blunts renal injury and MnSOD inactivation during I/R via stabilization of mitochondrial electron transport complexes. Our recent published studies show that the long-term (24 hr) pretreatment of rats with MnP significantly improved MnSOD activity and renal function during I/R (Appendix 2). New studies will determine whether MnP prevents mitochondrial superoxide production during ischemia by preserving the integrity of the mitochondrial electron transport complexes, hence maintaining normal mitochondrial ATP levels.